Intracellular Distribution of Manganese by the Trans-Golgi … · 2017. 11. 27. · Intracellular Distribution of Manganese by the TransGolgi Network - Transporter NRAMP2 is Critical

RESEARCH ARTICLE

Intracellular Distribution of Manganese by the Trans-Golgi Network Transporter NRAMP2 is Critical for Photosynthesis and Cellular Redox Homeostasis

Santiago Alejandro*, Rémy Cailliatte*, Carine Alcon, Léon Dirick, Frédéric Domergue1, David Correia, Loren Castaings, Jean-François Briat, Stéphane Mari and Catherine Curie2

BPMP, CNRS, INRA, Montpellier SupAgro, Univ Montpellier, Montpellier, France. 1Laboratoire de Biogénèse Membranaire CNRS - Université de Bordeaux - UMR 5200, Villenave d’Ornon, France. * S.A. and R.C. contributed equally to this work2Corresponding Author: [email protected]

Short title: Intracellular distribution of Mn by NRAMP2

One sentence summary: The metal transporter NRAMP2 controls photosynthesis and antioxidant defenses by releasing manganese from the trans-Golgi network, thus building up a cytosolic pool that feeds downstream organelles.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Catherine Curie ([email protected])

ABSTRACT Plants require trace levels of manganese (Mn) for survival, as it is an essential cofactor in oxygen metabolism, especially O2 production via photosynthesis and the disposal of superoxide radicals. These processes occur in specialized organelles, requiring membrane- bound intracellular transporters to partition Mn between cell compartments. We identified an Arabidopsis thaliana member of the NRAMP family of divalent metal transporters, NRAMP2, which functions in the intracellular distribution of Mn. Two knockdown alleles of NRAMP2 showed decreased activity of photosystem II and increased oxidative stress under Mn-deficient conditions, yet total Mn content remained unchanged. At the subcellular level, these phenotypes were associated with a loss of Mn content in vacuoles and chloroplasts. NRAMP2 was able to rescue the mitochondrial yeast mutant mtm1∆. In plants, NRAMP2 is a resident protein of the trans Golgi network. NRAMP2 may act indirectly on downstream organelles by building up a cytosolic pool that is used to feed target compartments. Moreover, not only does the nramp2 mutant accumulate superoxide ions, but NRAMP2 can functionally replace cytosolic superoxide dismutase in yeast, indicating that the pool of Mn displaced by NRAMP2 is required for the detoxification of reactive oxygen species.

1 INTRODUCTION

Plant Cell Advance Publication. Published on November 27, 2017, doi:10.1105/tpc.17.00578

©2017 American Society of Plant Biologists. All Rights Reserved

mailto:[email protected]

2

2 Mn serves as a cofactor for an array of enzymes and pathways, the most important of which is

3 the antioxidant defense enzyme manganese superoxide dismutase (Mn SOD), which

4 dismutates superoxide anion into the less toxic hydrogen peroxide and oxygen in

5 mitochondria and peroxisomes (del Rio et al., 2003). In addition, photosynthetic organisms

6 use Mn in the reaction center of photosystem II (PSII), in which a four Mn-cluster coordinates

7 with the oxygen evolving complex to perform the water-splitting reaction (Nickelsen and

8 Rengstl, 2013). For these reasons, Mn deficiency in plants targets two essential cellular

9 functions: antioxidant capacity and photosynthetic activity. Although Mn is rarely limiting for

10 animal cells, its bioavailability to plants is dramatically reduced in dry, well-aerated and

11 alkaline soils, which negatively affects plant growth. Visual symptoms of Mn shortage are

12 limited to young leaves, which are chlorotic at moderate deficiency but develop necrosis at

13 extreme deficiency levels (Schmidt et al., 2016). Consequently, the agronomical

14 repercussions of Mn deficiency have been underestimated and the molecular mechanisms at

15 work in the response to Mn limitation have been little explored compared to those of other

16 essential metals such as Fe, Zn and Cu. Mn excess can also be detrimental to organisms. Mn

17 toxicity leads to a predisposition to genomic instability (Garcia-Rodriguez et al., 2012) and

18 causes a neurological syndrome in human called manganism, with symptoms resembling

19 those of Parkinson disease (Roth, 2006). In plants, Mn toxicity is widespread in acidic soils,

20 where it becomes more available and provokes quite diverse symptoms often characterized by

21 brown speckles containing oxidized Mn on mature leaves and/or interveinal chlorosis and

22 necrosis (Marschner et al., 1986).

23 Most species acquire Mn via transporters of the Natural Resistance-Associated

24 Macrophage protein (NRAMP) family. These divalent metal/proton symporters harbor a

25 broad range specificity for metals (Nevo and Nelson, 2006). In Arabidopsis thaliana and rice

26 (Oryza sativa), high-affinity Mn uptake in root depends on the presence of NRAMP1 and

27 NRAMP5, respectively, at the root surface. Loss-of-function of Arabidopsis NRAMP1 or rice

28 NRAMP5 dramatically decreases the tolerance of the plant to Mn deficiency (Cailliatte et al.,

29 2010; Ishimaru et al., 2012; Sasaki et al., 2012). These proteins are therefore true orthologs of

30 the NRAMP transporter Smf1p in yeast (Cohen et al., 2000). Moreover, in most species, Mn

31 is a substrate of the high affinity, root-surface-localized Fe uptake transporter IRT1, which

32 was shown in Arabidopsis to contribute to Mn acquisition when combined with the nramp1

33 mutation (Castaings et al., 2016).

34 Our understanding of the mechanisms of the intracellular distribution of Mn is still

35 fragmentary. In plants, trafficking of Mn across the membranes of major organelles is

3

36 mediated by transporters belonging to a variety of transporter families. In seeds, the

37 tonoplastic VIT1 transporter mediates import of Mn and Fe into the vacuole (Kim et al., 2006;

38 Momonoi et al., 2009; Zhang et al., 2012) from where these metals are remobilized in

39 Arabidopsis by the NRAMP transporters NRAMP3 and NRAMP4. By mediating Mn efflux

40 from the vacuole, these two highly homologous tonoplastic proteins help maintain efficient

41 PSII activity under Mn-limiting conditions (Lanquar et al., 2005; Lanquar et al., 2010). Mn

42 vacuolar transporters also include i) CAX2, a Ca2+/cation antiporter involved in the vacuolar

43 sequestration of Mn (Pittman et al., 2004); ii) ZIP1, a homolog of IRT1 located within the

44 tonoplast membrane of root stele cells, where it is thought to participate in the remobilization

45 of Mn from the vacuole to the cytosol and mediate radial movement of Mn in the root (Milner

46 et al., 2013); and iii) METAL TOLERANT PROTEIN8 members in rice (Os-MTP8.1) and

47 Arabidopsis (MTP8), which belong to the subgroup of cation diffusion facilitators (CDF)

48 involved in proton/Mn antiport that help protect the cell from Mn toxicity by targeting excess

49 cytosolic Mn to the vacuole (Eroglu et al., 2016). How Mn is transported into mitochondria is

50 currently unknown. A study in yeast (Saccharomyces cerevisiae) initially identified the

51 Mtm1p carrier as the Mn importer into the mitochondrial matrix because its inactivation led to

52 reduced Mn SOD activity and was rescued by supplying the cells with a large amount of Mn

53 (Luk et al., 2003). However, the function of Mtm1p was subsequently reassigned to the

54 control of mitochondrial Fe homeostasis via the import of an enzymatic cofactor that plays a

55 key role in heme biosynthesis (Whittaker et al., 2015). Because the yeast Mtm1p mutant

56 mtm1∆ accumulates Fe in its mitochondria, mis-metallation of SOD2 occurs, leading to its

57 inactivation unless the cells are treated with high levels of Mn (Yang et al., 2006). At-MTM1,

58 a likely ortholog of Mtm1p, has been identified in Arabidopsis (Su et al., 2007). No Mn

59 transporter has yet been identified on the chloroplast envelope, but a thylakoid transporter has

60 recently been reported. PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is required

61 for the incorporation of Mn into the oxygen evolving complex of PSII, presumably by

62 transporting Mn into the thylakoid lumen (Schneider et al., 2016).

63 Mn trafficking into the secretory pathway appears to fulfill important functions in

64 maintaining Mn homeostasis in all kingdoms. Members of the P2A-type ATPase family were

65 shown to function as Ca2+/Mn2+ pumps in several organisms. Among these, yeast Pmr1p

66 transports Mn into Golgi-like compartments and its inactivation leads to Mn accumulation in

67 the cytosol, causing Mn toxicity (Rudolph et al., 1989). Two Pmr1p homologs in Arabidopsis,

68 ECA1 and ECA3, which are located in the endoplasmic reticulum (ER) and Golgi,

69 respectively, can rescue pmr1∆ growth defect under Mn stress. Inactivation of ECA1 and

4

70 ECA3 alters the tolerance of plants to suboptimal Mn conditions (Wu et al., 2002; Mills et al.,

71 2008). Moreover, MTP11, another Mn-CDF member from Arabidopsis, and its barley

72 homolog Hv-MTP8, are localized in Golgi /PVC compartments where they control plant

73 tolerance to high Mn through a mechanism that may involve vesicular trafficking to the

74 vacuole and/or secretion to the extracellular space (Delhaize et al., 2007; Peiter et al., 2007;

75 Pedas et al., 2014). Finally, Smf2p, an NRAMP family transporter, is also present in the

76 secretory pathway in yeast, although its target compartment (reported as being intracellular

77 vesicles) still awaits formal identification (Luk and Culotta, 20011). Since Smf2p is required

78 for the activity of Sod2p and protein glycosylation, two enzymes that require a Mn cofactor,

79 this transporter was proposed to function in feeding Mn to downstream organelles (Luk and

80 Culotta, 2001).

81 Among the six NRAMP family members in Arabidopsis, NRAMP1, NRAMP3 and

82 NRAMP4 have been assigned a function in Mn or Mn/Fe transport (Curie et al., 2000;

83 Thomine et al., 2000; Lanquar et al., 2005; Cailliatte et al., 2010; Lanquar et al., 2010;

84 Castaings et al., 2016). A fourth member, NRAMP6, has been described as being able to

85 transport Cd, but its physiological function is currently unclear (Cailliatte et al., 2009). In this

86 study, through reverse genetics and functional complementation of yeast mutants, we show

87 that NRAMP2 is an important player in the Mn nutritional pathway in Arabidopsis. Its

88 presence in the endomembrane system is required for adequate distribution of Mn between

89 cell compartments, which is crucial for optimal PSII activity and for controlling reactive

90 oxygen species (ROS) production.

91

92

93 RESULTS

94

95 NRAMP2 knock-down mutants are hypersensitive to Mn deficiency

96 To investigate the role of NRAMP2 in Arabidopsis, we used a reverse genetics

97 approach and analyzed a T-DNA insertion mutant line named nramp2-3 (Figure 1). NRAMP2

98 transcript levels in the mutant line, as measured by quantitative RT-PCR, were strongly

99 reduced but still retained at 5 to 10% of wild-type levels, indicating that nramp2-3 is a weak

100 allele of NRAMP2 (Figure 1A,B). A second mutant allele of NRAMP2, named nramp2-4,

101 showed 40% residual NRAMP2 transcript levels (Figure 1A,B). Therefore, both nramp2-3 and

102 nramp2-4 are knock-down alleles of NRAMP2. Since NRAMP family members have been

103 selected throughout evolution to transport Fe, Mn, or both, we searched for Fe and/or Mn

5

104 homeostasis defects in the nramp2 mutants. Fe-limited or excess conditions did not affect the

105 growth of the nramp2 mutants. In contrast, Mn limitation significantly altered the growth of

106 nramp2-3, and to a lesser extent that of nramp2-4 (Figure 1C, Supplemental Figure 1A,B),

107 affecting the root more than the shoot (Figures 1D,E). Mn-deficient nramp2-3 plants harbored

108 moderately chlorotic leaves (Figure 1C, Supplemental Figure 1A,B,D). Measurement of the

109 leaf SPAD index revealed a 44% reduction in chlorophyll content in Mn-deficient nramp2-3

110 plants (Supplemental Figure 1C). Mn addition to the hydroponic solution rescued the growth

111 defect of nramp2-3 and partially restored its chlorophyll content (Figure 1D,E; Supplemental

112 Figure 1C,D). Complemented nramp2 lines were generated by overexpressing a GFP-tagged

113 version of NRAMP2 under the control of the UBIQUITIN10 promoter (ProUB10:NRAMP2-

114 GFP) in the nramp2-3 mutant background (Figure 1B). Two of the complemented lines

115 obtained, named OxNR2 #3 and OxNR2 #6, which overexpressed NRAMP2 12- and 8-fold,

116 respectively (Figure 1B), showed full reversion of the nramp2-3 phenotype in Mn-deficient

117 growth conditions (Figure 1C-E). Altogether, these data demonstrate that decreasing

118 NRAMP2 expression results in hypersensitivity of the plant to Mn deficiency, which suggests

119 that NRAMP2 plays a role in Mn homeostasis.

120 We next measured Mn contents in shoots and roots of the nramp2-3 mutant and

121 complemented lines (Figure 2). Under Mn replete conditions, both mutant and overexpressor

122 lines accumulated significantly higher levels of Mn in shoots and roots than wild type

123 (Supplemental Table 1). Upon Mn shortage, Mn levels in the tissues of the mutant were not

124 reduced. Instead, the Mn content increased by 26% in shoots of the nramp2-3 mutant, a defect

125 that was fully rescued in the two complemented lines (Figure 2, Supplemental Table 1). This

126 result therefore ruled out the possibility that NRAMP2 functions in the acquisition of Mn

127 from the growth medium. We next tested whether an increase in NRAMP1 expression in roots

128 could account for the high level of Mn in the nramp2-3 mutant subjected to Mn deficiency.

129 The accumulation of NRAMP1 transcripts, however, was not significantly modified in

130 nramp2-3 roots in response to Mn-deficiency (Supplemental Figure 2).

131

132 NRAMP2 expression is ubiquitous in the plant

133 We previously showed that NRAMP2 is expressed in roots and to a lesser extent in shoots

134 (Curie et al., 2000). To examine the response of NRAMP2 to Mn nutrition, plants were grown

135 in hydroponic conditions for 4 weeks including, or not, a 3-week period of Mn deficiency

136 (Figure 3). The results confirmed the higher accumulation of NRAMP2 transcripts in roots

6

137 (Figure 3A). Moreover, the expression of NRAMP2 was induced approximately 2.5-fold in

138 response to Mn deficiency both in roots and shoots compared to the control (Figure 3A).

139 To assess more precisely the localization of NRAMP2 expression in plant tissues, we

140 generated transgenic plants carrying the ß-glucuronidase (GUS) reporter gene under the

141 control of the NRAMP2 promoter. Histochemical analysis of GUS activity showed staining in

142 vascular tissues of roots, cotyledons and leaves as well as in trichomes of young aerial tissues

143 (Figure 3B, panels a-c). In roots, staining was restricted to the stele, initiating in the

144 elongation zone and strengthening in older parts of the root (Figure 3B, panel d). Cross

145 sections through the root revealed preferential expression in the pericycle (Figure 3B, panel

146 e). In addition, transcriptional profiling in isolated root cell layers (Dinneny et al., 2008) also

147 revealed lower but significant NRAMP2 expression in epidermis and cortex, particularly close

148 to the root tip (http://www.bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi).

149

150 NRAMP2 is localized in the TGN compartment

151 In order to identify the target membrane of NRAMP2, we investigated the localization of

152 NRAMP2-GFP in complemented lines OxNR2 #3 and #6 expressing ProUB10:NRAMP2-

153 GFP (Figure 4). GFP fluorescence, as observed by confocal imaging of root cells of 5-day-old

154 plants, appeared as dot-like structures throughout the cytoplasm, with no labeling of the

155 plasma membrane, which was exclusively labeled with the endocytic tracer FM4-64 (Figure

156 4A). This fluorescence pattern was unchanged under different Mn regimes. Kinetic analysis

157 of the uptake of FM4-64 revealed partial co-localization of FM4-64 with NRAMP2-GFP after

158 15 min of treatment (Figure 4B), thus indicating that NRAMP2 localizes to the

159 endomembrane system. To identify the compartment labeled by NRAMP2-GFP,

160 ProUB10:NRAMP2-GFP was co-expressed with a set of organelle markers either by transient

161 expression in tobacco (Nicotiana tabacum) or through outcrossing with stably transformed

162 Arabidopsis marker lines. The two expression systems produced an identical pattern of

163 fluorescence for NRAMP2-GFP (Supplemental Figure 3A,B). No significant co-localization

164 was observed between NRAMP2-GFP and the peroxisomal marker px-rk CD3-983

165 (Supplemental Figure 3C,F). NRAMP2-GFP co-localized partially with the Golgi marker

166 SYP32 (Supplemental Figure 3D,G), and with the endosomal marker Rab A1g (Supplemental

167 Figure 3E,H).

168 To narrow down the localization of NRAMP2 within the endomembrane system,

169 ProUB10:NRAMP2-GFP was co-expressed with markers of subcompartments of the Golgi

170 apparatus in tobacco leaves. NRAMP2-GFP was in all instances closely associated, - but

http://www.bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi)

7

171 never co-localized -, with cis- and trans-Golgi markers ERD2 and Sialyl transferase (ST),

172 respectively (Figure 4C). Since NRAMP2-GFP was systematically distal to the ST-RFP trans-

173 Golgi marker (Figure 4C), we suspected that it was localized to the trans-Golgi Network

174 (TGN) compartment. Indeed, NRAMP2-GFP showed near-perfect co-localization with the

175 TGN marker SYP61-RFP (Figure 4D). Pearson correlation coefficients and Mander’s overlap

176 coefficients above thresholds were calculated for NRAMP2-GFP with either the marker

177 ERD2-CFP, ST-RFP or RFP-SYP61 and confirmed the overlap between NRAMP2 and

178 SYP61 proteins (Supplemental Table 2). Therefore, NRAMP2 is a protein of the endosomal

179 network predominantly located in the TGN.

180

181 Characterization of the role of NRAMP2 in Mn transport by heterologous expression

182 analysis in yeast

183 To obtain insight into the Mn transport activity of NRAMP2, we tested its ability to

184 functionally complement yeast mutants inactivated in various Mn transport systems (Figure

185 5). The yeast NRAMP homolog Smf1p, which mediates Mn uptake at the plasma membrane

186 (Supek et al., 1996), was investigated first. Growth of the smf1∆ mutant, which is reduced in

187 the presence of EGTA due to Mn limitation, was not restored by the expression of

188 Arabidopsis NRAMP2 (Figure 5A), unlike what was observed with Arabidopsis NRAMP1

189 (Curie et al., 2000; Thomine et al., 2000). We also previously showed that unlike NRAMP1,

190 NRAMP2 did not complement the plasma membrane iron uptake defect of the fet3∆fet4∆

191 mutant (Curie et al., 2000). Therefore, NRAMP2 is unlikely to perform uptake at the plasma

192 membrane, a result that is consistent with our finding that NRAMP2 is located in an

193 intracellular compartment and that Mn acquisition is not affected in the nramp2 mutant. Since

194 Smf2p in yeast had been described as an endomembrane Mn transporter (Cohen et al., 2000),

195 we tested whether NRAMP2 could functionally replace Smf2p. Growth of smf2∆, which like

196 smf1∆ is reduced in the presence of EGTA, was fully restored by the expression of NRAMP2

197 (Figure 5A). Phenotypes of smf2∆ also include a strong decrease in cellular Mn content and

198 Mn SOD activity (Luk and Culotta, 2001). As expected, NRAMP2 restored both the Mn

199 content and Mn SOD activity of smf2∆ (Figure 5B,C), suggesting that NRAMP2 is a true

200 ortholog of the yeast Smf2p intracellular Mn transporter.

201 We also tested the capacity of NRAMP2 to rescue pmr1∆, a yeast mutant lacking the

202 Mn transport ATPase for the Golgi. pmr1∆ is hypersensitive to high Mn due to increased Mn

203 accumulation in the cytosol. The expression of NRAMP2 did not rescue the pmr1∆ phenotype

204 (Figure 5E). This finding suggests that NRAMP2 activity does not reduce cytosolic Mn

8

205 concentrations, which in turn implies that it mediates Mn import into the cytosol.

206 Interestingly, we found that NRAMP2 also efficiently rescued the sensitivity of mtm1∆ to

207 EGTA (Figure 5D). mtm1∆ was shown to misincorporate Fe instead of Mn into mitochondrial

208 SOD and can be rescued by supplying excess Mn to the cell (Yang et al., 2006; Whittaker et

209 al., 2015). Since NRAMP2 is a protein in the TGN membrane, its ability to rescue mtm1∆

210 supports a role for this protein in improving cellular Mn availability.

211

212 PSII activity is reduced in the nramp2-3 mutant exposed to Mn starvation

213 Since a cluster of four Mn atoms is associated with the oxygen-evolving complex of PSII, we

214 searched for defects of photosynthesis activity in the nramp2-3 mutant that could explain its

215 slow growth under Mn-deficiency. We measured a number of photosynthesis parameters,

216 including maximal photochemical efficiency of PSII (Fv/Fm) and PSII operating efficiency

217 (ΦPSII). The Fv/Fm values (close to 0.8) were unchanged for all genotypes grown under

218 control conditions (Figure 6, left panel). Under Mn limitation, the Fv/Fm value decreased by

219 30% in wild-type plants, thereby highlighting the requirement of Mn for PSII activity, and

220 noticeably dropped by an additional 10% in leaves of the nramp2-3 (Figure, 6 right panel,

221 Supplemental Table 3) and nramp2-4 mutants (Supplemental Table 3). This phenotype was

222 rescued in the two OxNR2-complemented lines (Figure 6, right panel, Supplemental Table 3).

223 Thus, NRAMP2 is required to maintain optimal PSII activity in conditions of limited Mn

224 availability. Electron transfer flow downstream of PSII, which is estimated by the ΦPSII value,

225 was also more dramatically reduced under Mn deficient conditions in the two nramp2 mutants

226 compared to wild-type plants and was restored to a higher value than wild type in the OxNR2

227 complemented lines (Supplemental Table 3). Therefore, photosynthesis efficiency under Mn

228 deficient conditions requires an active NRAMP2.

229

230 The growth defect of the nramp2 mutant is associated with reduced Mn levels in

231 chloroplasts and vacuoles

232 To refine the role of NRAMP2 in the mobilization of internal Mn pools, Mn content was

233 measured in protoplasts and subcellular fractions of wild type, nramp2-3 and the OxNR2 #3

234 complemented line grown in either Mn-replete or Mn-deficient conditions (Figure 7 and

235 Supplemental Table 4). In Mn-starved nramp2-3 plants that display a strong phenotype, total

236 Mn content remained unchanged in protoplasts compared to protoplasts isolated from wild-

237 type plants (Figure 7A). This confirmed that inactivation of NRAMP2 affects the intracellular

238 distribution of Mn rather than total cellular content. We next tested whether the reduced

9

239 photosynthesis activity measured in nramp2-3 was associated with a lower Mn content in the

240 chloroplasts. Under Mn deficiency, the Mn concentration dropped almost two-fold in the

241 chloroplasts of nramp2-3 relative to wild-type plants, a defect that was rescued in the OxNR2

242 #3 line (Figure 7B). This suggested that reduced PSII activity in Mn-deficient nramp2-3

243 plants is caused by the lesser availability of Mn in the chloroplasts. Vacuoles were also

244 isolated from the same protoplasts in order to test for a general defect of Mn content in

245 organelles. Indeed, nramp2-3 vacuoles showed reduced Mn concentration (Figure 7C),

246 confirming that NRAMP2 is required to maintain optimal Mn concentration in several

247 organelles. We therefore concluded that under Mn deficiency, NRAMP2 contributes to

248 building up an available pool of cellular Mn to be used by downstream compartments

249 including - but most likely not restricted to- chloroplasts and vacuoles.

250

251 Cellular redox homeostasis is disturbed in the nramp2 mutant

252 The second prime target of Mn deficiency in plants is the mitochondrial superoxide

253 dismutase, or Mn SOD, the function of which is affected in the yeast smf2∆ mutant (Liu and

254 Culotta, 2001; this work). To test whether Mn SOD activity is affected in the nramp2 mutant,

255 an in-gel SOD activity assay was performed (Figure 8). Despite the prolonged Mn starvation

256 treatment imposed in this experiment, Mn SOD activity remained unaffected regardless of the

257 genotype (Figure 8A). These data indicate that in contrast to PSII, Mn SOD activity is

258 maintained in the nramp2 mutant under Mn deficient conditions. The most striking change

259 was observed for Cu/Zn SOD, the activity of which was strongly enhanced in nramp2 plants

260 grown in Mn-limiting conditions (Figure 8A). Complementation of the mutant with NRAMP2

261 efficiently normalized the activity of Cu/Zn SOD (Figure 8A). A slight increase in Fe SOD

262 activity was also observed in Mn-deficient nramp2-3 plants; contrary to Cu/Zn SOD,

263 however, Fe SOD activity was not rescued in the two OxNR2 complemented lines (Figure

264 8A).

265 The enhanced activity of Cu/Zn SOD revealed an imbalance of the redox status in Mn-

266 deficient nramp2 cells. We thus monitored the production of reactive oxygen species (ROS)

267 in the nramp2 mutant, especially the superoxide anion (O2.-), which is the substrate of Cu/Zn

268 SOD. In situ detection of O2.- ions was carried out by staining mature leaves with nitroblue

269 tetrazolium (NBT). Leaves of the nramp2-3 mutant showed strong purple NBT staining

270 regardless of the Mn regime, a phenotype that was mostly reverted in the OxNR2 #3

271 complemented line (Figure 8B). Increased NBT staining was not observed in the nramp3

272 nramp4 double mutant (Figure 8B), even though this mutant shared several Mn-related

10

273 phenotypes with nramp2 when exposed to Mn deficiency, including a growth defect and an

274 alteration of Mn homeostasis in chloroplasts (Lanquar et al., 2010).

275 In parallel, we tested whether NRAMP2 could rescue the oxidative stress of the sod1∆

276 yeast mutant defective in cytosolic Cu/Zn SOD. sod1∆ showed sensitivity to as low as 1 mM

277 EGTA, a phenotype that was fully rescued by the expression of NRAMP2 (Figure 8C). This

278 indicated that the pool of Mn displaced by NRAMP2 can efficiently replace cytosolic SOD

279 activity in yeast. Altogether, these results support the view that the Mn transport activity of

280 NRAMP2 takes part in the cellular antioxidant pathway.

281

282 Leaf surface permeability is affected in the nramp2-3 mutant

283 Mn-starved nramp2-3 plants displayed somewhat low turgor in leaves. A relationship

284 between tissue Mn content and cuticular wax synthesis in leaves has previously been pointed

285 out but is not well documented (Wilson et al., 1982; Hebbern et al., 2009). We therefore

286 investigated the permeability of the leaf surface in the nramp2-3 mutant under Mn-replete or

287 deficient conditions (Figure 9). The integrity of the leaf cuticle can be revealed by staining

288 with an aqueous solution of the hydrophilic dye toluidine blue (TB) (Tanaka et al., 2004). TB-

289 exposed rosette leaves of the nramp2-3 mutant showed an irregular punctuate staining after 10

290 minutes that contrasted with the intact cuticles of wild type and OxNR2 #3 leaves, suggesting

291 that the cuticles are permeable in nramp2-3 leaves (Figure 9A). By contrast, the nramp1-1

292 mutant, whose Mn content is strongly reduced under Mn deficiency (Cailliatte et al., 2010),

293 showed the same intensity of TB staining as wild-type leaves (Supplemental Figure 4). This

294 suggested that, rather than Mn deficiency, it is the adequate allocation of Mn in cell

295 compartments that is crucial for producing leaf surface components. Finally, we measured the

296 cutin content in the leaves of nramp2-3 and NRAMP2-complemented line OxNR2 #3 (Figure

297 9B, Supplemental File 1). Upon Mn deficiency, the cutin load was reduced by 40% in wild-

298 type plants, indicating that Mn is required for the production of an intact cuticle in

299 Arabidopsis leaves. Cutin content was unchanged in the nramp2-3 mutant in control condition

300 but when exposed to Mn deficiency, nramp2-3 was more severely affected than wild-type

301 plants, showing a more than 70% decrease. Consistent with TB staining, this phenotype of

302 nramp2-3 in Mn-deficient conditions was rescued in the OxNR2 #3 complemented line.

303 Taken together, these results indicate that Mn compartmentalization by NRAMP2 plays an

304 important role in assembling the leaf cuticle in Arabidopsis.

305

11

306 Genetic interaction between NRAMP2 and the tonoplastic NRAMP members NRAMP3

307 and NRAMP4

308 Hypersensitivity to Mn deficiency, reduced Mn content in chloroplasts and decreased

309 photosynthesis activity are features that are reminiscent of the phenotype of the nramp3

310 nramp4 double mutant. We therefore tested functional redundancy between NRAMP2 on one

311 hand, and NRAMP3 and NRAMP4 on the other hand, by crossing the nramp2 and nramp3

312 nramp4 mutants. The triple nramp2 nramp3 nramp4 mutant did not exhibit higher sensitivity

313 to Mn deficiency than the two parental lines, as shown by the equivalent reduction in

314 photosynthetic activity in all of these mutants (Supplemental Table 5). This suggested that the

315 three transporters act in the same pathway to provide Mn to the chloroplasts. Furthermore,

316 superoxide production was enhanced in the nramp2 nramp3 nramp4 triple mutant, as

317 observed in nramp2 (albeit seemingly less so) but not in nramp3 nramp4, thus indicating that

318 NRAMP2 is epistatic to NRAMP3/NRAMP4 (Supplemental Figure 5).

319

320

321 DISCUSSION

322 In this study, we characterized the phenotypic changes in Arabidopsis plants carrying a

323 knockdown allele of NRAMP2. NRAMP2 is a protein localized in the TGN membrane that

324 can functionally replace the yeast endomembrane Mn transporter Smf2p. The nramp2 mutant

325 displays pleiotropic defects that are exacerbated in Mn-deficient conditions and that

326 collectively point to a role of the NRAMP2 transporter in the intracellular distribution of Mn

327 to its target compartments.

328

329 Mn is a physiological substrate for NRAMP2

330 Our study provides strong, although indirect, arguments in favor of NRAMP2 acting as an Mn

331 transporter. The strongest argument is its ability to rescue the growth of a yeast mutant with

332 an inactivated SMF2 gene, which encodes an NRAMP homolog with reported Mn transport

333 activity. NRAMP2 can fully substitute for Smf2p, since its expression not only restored the

334 growth of the smf2∆ mutant in Mn-limiting conditions, but it also rescued its defective

335 MnSOD activity, suggesting that NRAMP2 is a true ortholog of yeast Smf2p. In addition, we

336 obtained evidence that NRAMP2 functions as a Mn transporter in planta. We found that

337 NRAMP2 knockdown plants exhibited impaired growth, specifically in Mn-limiting

338 conditions, whereas NRAMP2 overexpressing lines show hypertolerance to this treatment.

339 Furthermore, the Mn content in two cellular compartments, vacuoles and chloroplasts, was

12

340 markedly reduced in the nramp2 mutant. Lastly, NRAMP2 gene expression was induced in

341 response to Mn deficiency, which supports its involvement in Mn homeostasis. Thus, out of

342 the six NRAMP transporters in Arabidopsis, NRAMP2 represents the fourth member to play a

343 major role in Mn transport, next to NRAMP1, NRAMP3 and NRAMP4. The same

344 observation holds true for the characterized rice Os-NRAMP members and might be a general

345 feature of plant NRAMPs, thus distinguishing them from animal NRAMPs, which appear to

346 be more active in Fe transport.

347

348 NRAMP2 is a resident TGN protein

349 Unlike NRAMP1, NRAMP2 failed to rescue the plasma membrane Mn uptake of the yeast

350 ∆smf1 mutant but successfully substituted for Mn transport by Smf2p, which is located on

351 vesicles of the yeast endomembrane system (Portnoy et al., 2000; Luk and Culotta, 2001). We

352 therefore made the assumption that NRAMP2 works intracellularly in the plant. In plant cells,

353 we found that NRAMP2 localized to a vesicular compartment. Using a set of endomembrane

354 markers, we showed that NRAMP2 primarily colocalized with the TGN marker SYP61. Since

355 endocytosed plasma membrane proteins are initially targeted to early endosomes (EE), and

356 since TGN can also function as an EE in plants (Uemura and Nakano, 2013), a possibility

357 existed that NRAMP2 was in fact cycling between the plasma membrane and TGN.

358 Interestingly, dual targeting of NRAMP1 between the plasma membrane and intracellular

359 vesicles of the endomembrane pathway was recently reported (Agorio et al., 2017). Unlike

360 NRAMP1, however, we did not find evidence for the presence of NRAMP2 at the cell

361 surface, regardless of whether NRAMP2-GFP-expressing plants were Mn-starved or treated

362 with the endocytosis inhibitor Tyrphostin A23. Consistent with our localization data,

363 NRAMP2, but none of the other Arabidopsis NRAMP isoforms, was identified in a proteomic

364 study performed on affinity purified SYP61-containing TGN vesicles (Drakakaki et al.,

365 2012). Therefore, NRAMP2 is likely a resident TGN protein.

366

367 Loss of NRAMP2 affects Mn content in organelles

368 Our data reveal that PSII activity is severely reduced in leaves of the nramp2 mutant

369 exposed to Mn deficiency and that this decrease correlates with reduced Mn content in the

370 chloroplasts. This strongly suggests that NRAMP2 helps maintain chloroplastic Mn at an

371 optimal concentration for photosynthetic activity. We established that in addition to

372 chloroplast content, vacuolar Mn content is also significantly reduced in the nramp2 mutant.

373 Thus, rather than specifically targeting the chloroplast, we suspected that NRAMP2 works

13

374 upstream of several organelles, probably by functioning at an early crossroad in the

375 distribution pathway. If this was indeed the case, we reasoned that NRAMP2 would likely

376 distribute Mn to most of its target organelles. Consistent with this idea, we found that

377 NRAMP2 complemented the defective growth of the mitochondrial mutant mtm1∆ under Mn

378 deficiency, suggesting that NRAMP2 increases Mn availability for this organelle. In

379 Arabidopsis, however, Mn SOD activity, which we used as a proxy for mitochondrial Mn

380 level, remained unchanged in the nramp2 mutant, regardless of the Mn regime. This suggests

381 that Mn levels in mitochondria do not rely on the Mn transport activity of NRAMP2 or that an

382 alternative pathway exists that compensates for the absence of NRAMP2. Interestingly, Mn

383 SOD activity in wild-type plants was unaffected by Mn starvation, whereas the same

384 conditions efficiently depleted chloroplast content and affected PSII activity. A similar result

385 was previously reported (Lanquar et al., 2010). This suggests the existence of a hierarchy of

386 Mn allocation to the organelles in which maintaining mitochondrial Mn homeostasis is a

387 priority for the plant. Exactly the opposite result was obtained in Chlamydomonas reinhardtii,

388 where loss of activity of Mn SOD precedes the loss of PSII activity (Allen et al., 2007). In

389 conclusion, such a strong mechanism of preservation of Mn SOD activity in Mn-starved

390 Arabidopsis plants likely masks a potential impact of NRAMP2 mutation on mitochondrial

391 Mn content.

392 The chloroplastic pool of Mn was previously proposed to originate from the vacuole

393 because mutating the tonoplastic Mn transporters NRAMP3 and NRAMP4, which remobilize

394 stored Mn from the vacuoles, led to a decreased concentration of Mn in chloroplasts (Lanquar

395 et al., 2010). The finding that metal content in chloroplasts is tightly linked to the capacity of

396 metal release from the vacuole was further demonstrated in two independent studies. When

397 both the Arabidopsis YSL4 and YSL6 chloroplast transporters were inactivated, which led to

398 increased Fe accumulation in the chloroplasts, the expression of NRAMP3 and NRAMP4 was

399 dramatically inhibited, presumably as a feed-back mechanism aimed at preventing Fe

400 overload into the chloroplasts (Divol et al., 2013). Furthermore, the steady state level of

401 ferritins, the stability of which depends on the presence of iron in the chloroplast, is

402 drastically reduced in the nramp3 nramp4 double mutant background (Ravet et al., 2009).

403 Again, these results suggest that a direct link exists between Fe efflux from the vacuole and

404 Fe content in the chloroplasts. Altogether, these studies indicate that at least a large portion of

405 these metals contained in the chloroplasts originates from the vacuoles. Within the pathway

406 that provides Mn to the chloroplasts, several lines of evidence support the idea that the three

407 transporters, NRAMP2, NRAMP3 and NRAMP4, act together, with NRAMP2 operating

14

408 before the other two: i) The phenotype of hypersensitivity to Mn deficiency of the nramp2

409 nramp3 nramp4 triple mutant is similar in intensity to that of the nramp2 single mutant; ii)

410 The pleiotropic phenotypes of nramp2 include all of the alterations of nramp3 nramp4 but

411 also additional ones such as oxidative stress imbalance, vacuolar Mn depletion and cuticular

412 wax defects; and iii) NRAMP2, NRAMP3 and NRAMP4 have overlapping spatial expression

413 patterns and respond similarly to Mn deficiency. Together, these experiments argue against

414 the direct uptake of Mn from the cytosol and rather favor a strategy by which Mn first

415 accumulates into the vacuole prior to being dispatched to its target organelles. Favoring safe

416 metal storage into the vacuole, from which at least the chloroplast draws part of its supply,

417 may be a strategy used by the plant to prevent toxicity of these reactive metals in the cytosol.

418

419 Mn and leaf surface permeability

420 We demonstrated that leaf surface permeability and cutin production are disturbed specifically

421 in nramp2-3 plants exposed to Mn deficiency and more generally as a result of Mn starvation.

422 A reduction in the epicuticular layer in response to Mn deficiency was previously described in

423 barley, where it was shown to be associated with a change in transpiration rate and water use

424 efficiency (Hebbern et al., 2009). This transformation, which may partly explain the reported

425 susceptibility of Mn-deficient crops to pathogens, might correspond to an adaptation of the

426 plant to reduced PSII activity. Furthermore, Mn availability was also found to regulate root

427 suberization, resulting in higher permeability of the stele in response to metal shortage

428 (Barberon et al., 2016). Interestingly, whereas both nramp1 and nramp2 are Mn-starved under

429 conditions of low Mn supply (Cailliatte et al., 2010), only the leaf permeability of nramp2

430 was more affected than wild-type plants in these experiments. Thus, it appears as though this

431 phenotype is caused by Mn deficiency in a specific cell compartment rather than by global

432 plant Mn status. Cuticle components are synthesized predominantly in the endoplasmic

433 reticulum (Yeats and Rose, 2013) and are then thought to be secreted to the cell wall via an

434 ER-Golgi route (Hoffmann-Benning and Kende, 1994). To date, no Mn-dependent enzymatic

435 activity has been identified in the biosynthetic pathway of cuticular components. As is the

436 case for the vacuole, chloroplast and mitochondria, the availability of Mn to endosomal

437 compartments such as the ER and Golgi apparatus might be reduced in the nramp2 mutant,

438 hence affecting a potential Mn-containing enzyme involved in cuticle biosynthesis. It is

439 noteworthy that an important class of Mn-containing proteins of the cell is constituted by the

440 large family of glycosyl transferases (GT), which are mainly located in the Golgi, where they

441 glycosylate various substrates, among which are important glycoproteins of the cell wall.

15

442 Whether NRAMP2 brings Mn to Golgi-localized GTs or ER-localized cuticle biosynthetic

443 enzymes deserves further investigation.

444

445 Oxidative stress level is elevated in nramp2

446 The nramp2 mutant experiences redox imbalance under Mn-deficient conditions based on

447 increased Cu/Zn SOD activity and enhanced O2- production. The high O2- level in the mutant

448 is likely not provoked by reduced mitochondrial Mn SOD activity, as we found no

449 modification of this activity in nramp2, thus ruling out mitochondria as a source of O2-. Since

450 we measured increased Cu/Zn SOD, we speculated that O2- radicals produced by nramp2

451 originate from defective photosynthesis. Yet, the nramp3 nramp4 double mutant, which also

452 displays reduced Mn content in chloroplasts and impaired Fv/Fm activity (Lanquar et al.,

453 2010), shows no defect in redox homeostasis. Therefore, the observation that only nramp2

454 plants showed higher oxidative stress levels strongly suggests that the localization of

455 NRAMP2 in the TGN, and therefore intracellular management of Mn pools, is central to the

456 cellular redox imbalance of the mutant.

457 In yeast, small molecular weight Mn complexes in the cytosol display SOD activity,

458 thereby acting as an alternative, parallel system to enzymatic SODs and providing an

459 explanation for the rescue of sod1∆ by Smf2p (McNaughton et al., 2010). We found in this

460 work that Arabidopsis NRAMP2 too can compensate for the absence of Sod1p in yeast. The

461 role of Mn-Pi or Mn2+ complexes with small organic molecules as potent scavengers of

462 superoxide has also been revealed in bacteria, and evidence that such antioxidant Mn species

463 exist in multicellular organisms was recently obtained in Caenorhabditis elegans and animal

464 sperm (Lin et al., 2006); reviewed in (Culotta and Daly, 2013). Thus, an appealing hypothesis,

465 which deserves future investigations, is that like its yeast ortholog, NRAMP2 could build up a

466 pool of cytosolic antioxidant Mn in Arabidopsis.

467

468 NRAMP2 is crucial for Mn nutrition upon Mn limitation

469 Within the cell, the TGN is a sorting hub for post-Golgi trafficking pathways involved in

470 secretory or vacuolar transport. The presence of NRAMP2 in the TGN suggests it plays a role

471 in the mobilization of Mn from, or into, these vesicles. Since proteins of the NRAMP family

472 use the proton gradient as a driving force (Nevo and Nelson, 2006), they are known to

473 transport metals from a more acidic to a less acidic compartment. Consequently, except for

474 the few studies that proposed that transport by NRAMP occurs in the direction of the exit of

475 metals from the cytosol (Techau et al., 2007),(Garcia-Rodriguez et al., 2015), the vast

16

476 majority of the reported NRAMP transporters, including all plant members, import metals

477 into the cytosol (Cailliatte et al., 2010). Following the proton cotransport dogma, the presence

478 of NRAMP2 on TGN vesicles, which are more acidic than the cytosol (Martiniere et al.,

479 2013), is consistent with NRAMP2 mediating Mn influx into the cytosol. In addition, the

480 failure of NRAMP2 to rescue the pmr1∆ yeast mutant defective in Golgi Mn uptake also

481 argues against an activity of NRAMP2 in transport toward the TGN lumen. Several

482 observations support the hypothesis that NRAMP2 releases Mn from the TGN into the

483 cytosol and hence contributes to the build up of a cytosolic Mn pool that would be used to

484 feed Mn both to downstream organelles and to the anti-oxidant machinery of the cell: i) the

485 Mn content in two organelles, vacuoles and chloroplasts, correlates with the expression level

486 of NRAMP2: vacuoles and chloroplasts of the nramp2 mutant both harbor low Mn content; ii)

487 in yeast, NRAMP2 expression rescues the mitochondrial mtm1∆ mutant, the growth of which

488 depends on high Mn supply to the mitochondria (Yang et al., 2006); these two arguments

489 strongly support a role for NRAMP2 in fueling the cytosolic Mn pool, which serves as a

490 source of Mn to the main organelles; iii) NRAMP2 is also able to functionally compensate for

491 the absence of cytosolic Sod1p in yeast, which we propose may occur through the

492 accumulation of cytosolic Mn antioxidant species having SOD activity. If a similar alternate

493 SOD system relying on Mn antioxidant species in the cytosol exists in plants, NRAMP2

494 represents a reasonable candidate to supply these from vesicular compartments.

495 Based on the phenotypes of an nramp2 knockdown allele and its combination with the

496 nramp3 and nramp4 mutations, we propose a model in which under Mn-limiting conditions, a

497 pool of Mn stored in the TGN is released by NRAMP2 and carried by a yet-to-be identified

498 pathway to the vacuole, from which it is secreted by NRAMP3 and NRAMP4 to provide a

499 significant portion of the Mn ions that reach the chloroplasts (Figure 10). In-depth analysis of

500 the cellular function of candidate Mn transporters, including members of the ECA, MTP and

501 NRAMP families, should identify additional important actors and shed further light into the

502 Mn trafficking pathway in plant cells, although chemical or genetically-encoded Mn sensors

503 will be needed to image Mn movement between cell compartments.

504

505

506 METHODS

507

508 Plant materials and growth conditions

17

509 Two Arabidopsis thaliana NRAMP2 mutant lines were used: nramp2-3 (unreferenced SALK

510 line kindly provided by Dr. Jose Alonso) and nramp2-4 (GK-306A04), both containing a T-

511 DNA inserted in the 5’-UTR of the NRAMP2 gene, at 68 base pairs and 43 base pairs

512 upstream of the ATG start codon, respectively. The nramp1-1 mutant was described

513 previously (Cailliatte et al., 2010). The nramp3-2 nramp4-2 double mutant was generated by

514 crossing SALK line 023049 (nramp3-2) with SALK line 003737 (nramp4-2).

515 Arabidopsis seedlings (ecotype Columbia-0) were grown in vitro under sterile conditions

516 at 21ºC with an 8h-light (composed of 50% sodium-vapor lamps and 50% metal halide (HQI)

517 lamps)/16h-dark cycle. Seeds were surface sterilized and sown on half-strength Murashige

518 and Skoog medium containing 1% sucrose, 2.5 mM MES and either 0.6% agar for horizontal

519 cultures or 1% agar for vertical cultures. The pH was adjusted to 5.7 with KOH.

520 For the hydroponic growth experiments, seeds were sown on vertical plates. 8-day-old

521 seedlings were placed in a modified Hoagland solution containing 0.25 mM NH4NO3, 1 mM

522 Ca(NO3)2, 0.2 mM KH2PO4, 1.4 mM KNO3, 0.4 mM MgSO4, 20 µM Fe(III)-Na-EDTA, 4.5

523 µM H3BO3, 0.03 µM CuSO4, 0.07 µM ZnSO4, and 0.02 mM MoO3. For control conditions,

524 the medium was supplemented with 5 µM MnSO4. The nutrient solution was renewed

525 weekly. The hydroponic cultures were grown in a controlled environment (8h-light 23ºC/16h-

526 dark cycles, 20ºC, 200 µE·s-1 light intensity, 70% relative humidity) for the amount of time

527 indicated in the figure legends.

528

529 Plasmid constructs

530 For tissue-specific expression studies, 983 bp of the promoter region located upstream of the

531 NRAMP2 initiation codon were amplified by PCR from BAC F8G22 using forward primers

532 OCC53 and OCC54 (see list of primers in Supplemental Table 6), introducing an XbaI and

533 NcoI site, respectively. The PCR product was cloned into the pBKS+-GUS vector. The

534 ProNRAMP2:GUS fusion cassette was then transferred into the XbaI site of the pGreen29

535 binary vector. For the ProUB10:NRAMP2-GFP fusion construct, the NRAMP2 cDNA was

536 amplified without its stop codon using primers NR2_ORF-F and NR2_ORF-R and cloned

537 into the pDONR 207 entry vector (Gateway, Invitrogen). The NRAMP2 ORF was then

538 subcloned into the pUBC-GFP-Dest vector (Grefen et al., 2010) to generate a C-terminal GFP

539 fusion. The GV3101 strain of Agrobacterium tumefaciens containing the ProNRAMP2:GUS

540 and ProUB10:NRAMP2-GFP constructs were transformed into Arabidopsis ecotype Col-0

541 wild-type and nramp2-3, respectively, by the floral dip method (Clough and Bent, 1998).

542

18

543 Cell fractionation

544 Chloroplasts isolation. Protoplasts were isolated by enzymatic digestion of approximately 3

545 g of leaves cut in 1-2 mm-wide stripes in 10 mL of MCP buffer (500 mM sorbitol, 1 mM

546 CaCl2, and 20 mM MES, adjusted to pH 6 with KOH) supplemented with 0.4% (w/v)

547 Macerozyme R10 (Duchefa Biochemie) and 0.8% (w/v) Cellulase Onozuka R10 (Duchefa

548 Biochemie) at 30ºC for 1 h 30 min. The protoplasts were filtered through a 75-mm nylon

549 mesh, pelleted (200g, 5 min, 4ºC), and washed twice with MCP buffer. The protoplasts were

550 diluted to 2x106 cells mL-1 and lysed by filtration through a polyester mesh (Spectrum, pore

551 size 21 µm). The lysed protoplasts were kept on ice. Percoll step gradient: bottom phase, 1

552 volume of 85% Percoll in washing medium (300 mM sorbitol, 40 mM Tricine pH 7.6, 2.5

553 mM EDTA, and 0.5 mM MgCl2); Top phase, 1 volume of 40% Percoll in washing medium.

554 Chloroplasts were isolated by loading the protoplast lysate on top of the Percoll step gradient

555 followed by centrifugation (2500g, 10 min, 4ºC, without brake) in a swinging rotor. Intact

556 chloroplasts were recovered by centrifugation at 200 g for 5 min at 4ºC (with no brake) from

557 the 40/85% Percoll interphase and washed two times with cold washing medium . The

558 integrity of the chloroplasts was verified by microscopy, and the number of chloroplasts was

559 estimated by counting with a Neubauer chamber.

560 Vacuole isolation. The vacuole isolation was carried out as previously described (Lanquar et

561 at., 2010) with some modifications. Briefly, the protoplasts were lysed by the addition of an

562 equal volume of protoplast lysis buffer (200 mM sorbitol, 10% [w/v] Ficoll 400, 20 mM

563 EDTA, 10 mM HEPES-KOH, pH 8, 0.15% [w/v] bovine serum albumin, and 2 mM

564 dithiothreitol [DTT] and 5 µg/ml neutral red) pre-warmed at 37ºC. Protoplast lysis was

565 monitored microscopically, and lysed protoplasts were kept on ice. Vacuoles were isolated

566 using a step gradient in swinging rotors (1,500g, 20 min, 4C): bottom phase: 1 volume of

567 lysed protoplasts; middle phase: 0.8 volume of lysis buffer diluted in vacuole buffer to a final

568 concentration of 4% (w/v) Ficoll; top phase: 0.2 volume of vacuole buffer (500 mM sorbitol,

569 10 mM HEPES pH 7.5 [KOH], 0.15% [w/v] bovine serum albumin and 4 mM DTT).

570 Vacuoles were recovered as a thin red layer at the interface between the middle phase and the

571 top phase. The purity of the vacuole preparation was monitored by microscopy and the

572 number of vacuoles was estimated by counting with a Neubauer chamber.

573

574 Gene expression analysis

575 Total RNA was extracted using the TRIZOL reagent (Invitrogen) following the

576 manufacturer’s instructions. Reverse transcription was performed with oligo d(T) primers on

19

577 1 to 2 µg of total RNA digested with DNAse I (Thermo Scientific) using a RevertAid First

578 Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s protocols.

579 The qPCRs were performed on the Roche Light Cycler 480 II instrument using the primer sets

580 listed in Supplemental Table 6 and SYBR® Premix Ex Taq (Tli RNaseH Plus), Bulk (Takara).

581

582 GUS histochemical analysis

583 Samples were vacuum infiltrated for 20 min and incubated for 18h in GUS buffer containing

584 50 mM NaPO4 pH 7,4, 2 mM ferrocyanide, 2 mM ferricyanide, 0.05% Triton X-100, and 1

585 mM X-Gluc (5-bromo-4-chloro-3-indolyl-!3-D-glucuronide). The samples were then soaked in

586 phosphate buffer (50 mM NaPO4 pH 7.2) and prefixed for 3 h in phosphate buffer containing

587 2% paraformaldehyde and 0.5% glutaraldehyde. The samples were cleared with successive

588 washes of increasing ethanol concentrations from 50 to 100% for direct observation or

589 embedded in 2-hydroxyethyl methacrylate (Technovit 7100; Heraus-Kulzer). Thin cross-

590 sections (5 µm) were cut using a Leica RM 2165 microtome, counter-stained with Schiff dye,

591 and observed under an Olympus BH2 microscope.

592

593 Yeast strains and growth conditions

594 Yeast cells were grown at 30ºC in YPD medium (2% glucose, 2% tryptone and 1% yeast

595 extract) or SD medium (2% glucose, 0,7% yeast nitrogen base with ammonium sulfate and 50

596 mM succinic acid pH5.5). Wild-type BY4741 (MATa; his3, leu2, met15, ura3); smf1Δ

597 (MATa; his3, leu2, met15, ura3, smf1::kanMX4), smf2Δ (MATa; his3, leu2, met15, ura3,

598 smf2::kanMX4), mtm1Δ (MATa; his3, leu2, met15, ura3, mtm1::kanMX4), pmr1Δ (MATa;

599 his3, leu2, met15, ura3, pmr1::kanMX4) and sod1∆

600 (MATa his3 ::1 leu2::0 met15::0 ura3::0 sod1::kanMX4) cells were transformed with

601 pYPGE15-NRAMP2 (Curie et al., 2000), where NRAMP2 is expressed under the control of

602 the constitutive high yeast phospho-glycerate kinase (PGK) promoter, or pYPGE15 empty

603 vector using electroporation. Yeast transformants were selected on medium lacking uracil.

604 For growth tests, yeast cells were first grown in liquid culture overnight at 30ºC in SD 605

medium. The cultures were adjusted to OD600 = 0.3 and aliquots of 5-fold serial dilutions were

606 plated on 2% (w/v) agar plates containing SD-Ura medium supplemented with EGTA

or 607 MnSO4. In the case of EGTA plates, SD medium was prepared with 0.7% YNB with low

608 metal concentration (US Biological, USA). To monitor the steady-state manganese levels

in 609 yeast cells, the strains were grown in 20 ml SD-Ura medium to an OD600 = 1.5, pelleted and

610 washed three times with 10 mM EDTA and twice with deionized water.

20

611

612 Superoxide dismutase activity

613 In yeast: Yeast cells were propagated in YPD medium to an OD600 of 1.5, harvested and

614 washed with cold water before being broken by agitation in the presence of glass-beads in

615 extraction buffer (100 mM NaPO4 pH 7.8, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1%

616 Triton X-100, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A and 1 µg/ml

617 leupeptin). The homogenate was centrifuged for 5 min at 2500g and the protein pellet was

618 solubilized in extraction buffer.

619 In plants: Leaves were ground in liquid nitrogen and resuspended in cold extraction buffer

620 (40 mM K2HPO4, 10 mM KH2PO4, 6 mM ascorbic acid, 0.05% !3-mercaptoethanol and 0.2%

621 Triton X-100) for 2 min, followed by 10 min centrifugation at 14,000 rpm at 4ºC.

622 Proteins were loaded onto a 12% non-denaturing PAGE gel and the SOD activity was

623 measured by nitroblue tetrazolium (NBT) staining. Briefly, the gel was soaked in NBT

624 solution (1 mg/ml) for 20 min in the dark and transferred into staining solution (33 mM

625 K2HPO4, 3.3 mM KH2PO4, 1 mg/ml riboflavin and 0.3% TEMED) for 20 min in the dark.

626 The gel was rinsed in distilled water before being exposed to high light until the bands

627 appeared.

628

629 Manganese content measurements

630 The roots were washed for 10 min with 10 mM EDTA and 5 min with 30 mM MgCl2,

631 followed by two rinses in deionized water. Shoots were simply rinsed twice with deionized

632 water. Plant samples and yeast pellets were dried at 80ºC for 4 days. For mineralization, the

633 tissues were digested completely in 50% HNO3 and 7.5% H2O2 using the Speedwave 2

634 microwave digestion system (Berghof, Germany). Mn measurements were performed by

635 atomic emission spectrometry (4200 MP-AES, Agilent).

636

637 Photosynthesis fluorescence measurements

638 A kinetic imaging fluorometer (FluorCam FC 800-O, Photon Systems Instruments, Czech

639 Republic) was used to capture fluorescence images and to estimate the maximal

640 photochemical efficiency of PSII [Fv/Fm = (Fm – Fo)/Fm] and effective photochemical

641 efficiency of PSII (ΦPSII) parameters. Control and Mn-free Arabidopsis plants were dark-

642 adapted (30 min), and all fluorescence measurements were performed in vivo at room

643 temperature. Saturating flashes (1000 µmol· m-2·s-1) were used to measure the maximum

644 fluorescence.

21

645

646 Chlorophyll measurements

647 A SPAD meter (SPAD-502Plus, Konica-Minolta, Japan) was used to take SPAD values from

648 two fully expanded young leaves on each plant. A total of six plants were measured for each

649 genotype and growth condition, and three SPAD values per leaf were averaged as the mean

650 SPAD value of the leaf.

651

652 Detection of superoxide radicals

653 O2- ions were visualized by staining leaves (leaf # 8-10) with nitroblue tetrazolium (NBT).

654 Briefly, excised leaves were vacuum infiltrated in NBT staining solution (1 mg/ml NBT, 10

655 mM Na2HPO4) for 20 min and incubated for 2-3 hours under the light. Chlorophyll was

656 removed by incubating the leaves for 5 min in boiling 96% ethanol. After cooling, the leaves

657 were immersed in water and photographed in 10% glycerol.

658

659 Permeability test with toluidine blue

660 Excised leaves were submerged into an aqueous solution of filtered 0.05% (w/v) TB in a

661 small Petri dish for the indicated amount of time. The leaves were gently washed with water

662 to remove excess TB.

663

664 Cutin load analysis

665 Three to five fully developed leaves were used per replicate. Freshly collected leaves were

666 scanned to measure their surfaces and immediately incubated in hot isopropanol for 30 min at

667 85°C. After cooling, the solvent was eliminated and the leaves were extensively delipidated

668 by extracting the soluble lipids successively with CHCl3:CH3OH (2:1, v/v), CHCl3:CH3OH

669 (1:1, v/v), CHCl3:CH3OH (1:2, v/v), and CH3OH. Each delipidation step was performed for

670 24 h at room temperature on a rotating wheel at 40 rpm. The delipidated leaves were dried in

671 a fume hood at room temperature for 2 days and then in a desiccator for another 2 days. Cutin

672 analysis was then performed as described previously (Domergue et al., 2010).

673

674 Confocal microscopy analysis

675 Nicotiana tabacum leaves were agro-infiltrated with the recombinant Agrobacterium

676 tumefaciens strains (strain GV3101, infiltration solution OD600= 0.1). After two days,

677 confocal imaging was performed using a Leica TCS SP8 laser scanning microscope with a

22

678 63X oil N.A. 1.4 immersion objective. Emission fluorescence of CFP, GFP and mCherry was

679 collected in line sequential scanning mode at 458 nm, 488 nm and 561 nm excitation,

680 respectively. The emitted signal was collected between 462-485 nm, 500-535 and 580-630 for

681 CFP, GFP and mCherry/RFP, respectively, and images were acquired with the best lateral and

682 axial resolution (pixel size 60/70 nm). Images were processed using ImageJ software. For the

683 FM4-64 endocytosis experiments, roots of 7-day-old Arabidopsis seedlings were incubated

684 for 5 min in 10 µM FM4-64 and rinsed with water at the time indicated in the figure legends.

685 The set of cell compartment markers (plasmids or Arabidopsis lines) used was as follows: Px-

686 rk983-mCherry (Nelson et al., 2002), Arabidopsis Wave lines (Geldner et al., 2009), ERD2-

687 CFP (Brandizzi et al., 2002), and SYP61-mRFP (Foresti et al., 2010).

688 Colocalization coefficients were computed with the JACoP plugin (rpearson, Pearson

689 correlation coefficient; M1 and M2, Mander’s overlap coefficients above threshold) (Bolte

690 and Cordelieres, 2006). In each experiment, 7 to 10 cells from the root elongation zone were

691 analyzed individually for each genotype.

692

693 Accession numbers

694 Sequence data from this article can be found in the Arabidopsis Genome Initiative or

695 GenBank/EMBL databases under accession numbers At1g80830 (NRAMP1), At1g47240

696 (NRAMP2), At2g23150 (NRAMP3), At5g67330 (NRAMP4), and At3g18780 (ACT2).

697

698 Supplemental Data

699 Supplemental Figure 1. nramp2 mutant alleles are chlorotic under Mn deficiency.

700 Supplemental Figure 2. Expression of NRAMP1 is not modified in the nramp2-3 mutant.

701 Supplemental Figure 3. Confocal microscopy observations of NRAMP2-GFP fluorescence

702 and colocalization with markers of various cell compartments.

703 Supplemental Figure 4. nramp2-3, but not the Mn-deficient mutant nramp1-1, shows leaf

704 permeability defects.

705 Supplemental Figure 5. Production of superoxide ions in the nramp2 nramp3 nramp4 triple

706 mutant.

707 Supplemental Table 1. Mn concentration (µg.g-1 DW) in rosette leaves and roots of WT,

708 nramp2-3 and the OxNR2 #3 and OxNR2 #6 complemented lines.

709 Supplemental Table 2. Quantitative analysis of NRAMP2-GFP colocalization with Golgi

710 markers.

23

711 Supplemental Table 3. Photosynthetic activity of nramp2 mutant alleles and the

712 complemented line OxNR2 #3.

713 Supplemental Table 4. Mn content is altered in vacuoles and chloroplasts of the nramp2-3

714 mutant.

715 Supplemental Table 5. Photosynthetic activity of single nramp2-3, double nramp3-2

716 nramp4-2 and triple nramp2-3 nramp3-2 nramp4-2 mutants.

717 Supplemental Table 6. List of the gene-specific primers used.

718 Supplemental File 1. ANOVA Table.

719

720

721 ACKNOWLEDGEMENTS

722 This work was funded by the Agence Nationale pour la Recherche through the ANR grants

723 PLANTMAN (ANR-2011-BSV6-004) and MANOMICS (ANR-2011-ISV6-001-01), by the

724 Centre National de la Recherche Scientifique and by the Institut National de la Recherche

725 Agronomique. Microscopy observations were made at the Montpellier RIO Imaging facility

726 (http://www.mri.cnrs.fr/). We thank Dr. Jose Alonso for providing nramp2-3 seeds. We are

727 indebted to Sandrine Chay and Brigitte Touraine for assistance with MP-AES and FluorCam

728 measurements, respectively. S.A.-M. and L.C. were supported by the Agence Nationale pour

729 la Recherche, and R.C. was supported by the French Ministry of Agriculture and Fisheries.

730

731 AUTHOR CONTRIBUTIONS

732 S.A., R.C., S.M. and C.C. designed the research. S.A., R.C., C.A., L.D., D.C., L.C., F.D.

733 performed research and analyzed data. C.A. performed confocal imaging experiments

734 and statistical analysis of colocalizations. F.D. performed lipid analyses. J.F.B.

735 contributed to discussions and constructive comments on the manuscript. The

736 manuscript was written by S.A. and C.C.

737

738 NOTE ADDED IN PROOF

739 While this manuscript was under revision, two mutant alleles of NRAMP2 (nramp2-1 and

740 nramp2-2) were reported [Gao, H., Xie, W., Yang, C., Xu, J., Li, J., Wang, H., Chen, X.,

741 and Huang, C.F. (2017) NRAMP2, a trans-Golgi network-localized manganese transporter,

742 is required for Arabidopsis root growth under manganese deficiency. New Phytologist doi:

743 10.1111/nph.14783].

744

http://www.mri.cnrs.fr/)

24

745

746

747 FIGURES LEGENDS

748 Figure 1. nramp2 mutants are hypersensitive to Mn deficiency.

749 (A) Position of the T-DNA insertion in two nramp2 mutant alleles relative to the ATG start

750 codon.

751 (B) Relative NRAMP2 transcript abundance measured by qRT-PCR in nramp2 mutant alleles

752 and in the two nramp2-3 complemented lines OxNR2 #3 and OxNR2 #6. Transcript levels are

753 expressed relative to those of the reference gene ACTIN ± SD (n=3 measurements per

754 sample). (C) Phenotypes of plants of the indicated genetic backgrounds grown for one week

755 in Mn-replete conditions (5 µM Mn SO4) followed by 3 additional weeks in Mn-free

756 hydroponic conditions.

757 (D) and (E) Shoot (D) and root (E) biomass production of the plants presented in C (-Mn) or

758 in Mn replete conditions (Control). Data are from one representative experiment (n = 10

759 plants). Panels D and E share color code legends. Mean ± SD. Asterisks indicate values

760 significantly different from those of wild type (Student’s t test, * P-value < 0.05, ** P-value <

761 0.01). Scale bars = 1 cm.

762

763 Figure 2. nramp2-3 shows Mn homeostasis defects under Mn-free conditions.

764 (A) Mn content in the Mn-deficient plants presented in Figure 1 and in control plants grown

765 in parallel in Mn-replete conditions shown in Supplemental Figure 1D. Data are from one

766 representative experiment (n = 4 plants). DW: Dry weight. Mean ± SD. Asterisks indicate

767 values significantly different from those of wild type (Student’s t test, * P-value < 0.01).

768

769 Figure 3. NRAMP2 expression analysis.

770 (A) Relative NRAMP2 transcript abundance in roots and shoots measured by qRT-PCR.

771 Transcript levels are expressed relative to those of the reference gene ACTIN ± SD (n=3

772 technical replicates). Wild-type plants were grown hydroponically for 3 weeks in control (5

773 µM MnSO4) or in Mn-free (-Mn) conditions. Data are from one representative experiment (n

774 = 3 plants). Mean ± SD. Asterisks indicate values significantly different from those of control

775 conditions (Student’s t test, * P-value < 0.01).

776 (B) Histochemical staining of GUS activity in proNRAMP2:GUS-transformed Arabidopsis

777 grown in Mn-replete conditions. (a) 6-day-old seedling. (b) Young rosette leaf. (c) Mature

778 rosette leaf. (d) Primary root of a one-week-old plant in the mature zone. (e) Root cross-

25

779 section in the mature zone. Ep: epidermis, Co: cortex, End: endodermis, Pe: pericycle. Data

780 are shown for one representative line out of 3 independent lines.

781

782 Figure 4. NRAMP2 is localized to the Trans-Golgi Network.

783 (A) and (B) Fluorescence pattern of the NRAMP2-GFP construct and kinetics of

784 colocalization with the endocytic tracer FM4-64 (A: 2 min, B: 15 min) observed by confocal

785 microscopy in roots of 5-day-old plants.

786 (C) and (D) Colocalization of NRAMP2-GFP with markers for cis-Golgi (ERD2-CFP), trans-

787 Golgi (ST-RFP), and TGN (SYP61-GFP) transiently co-expressed in tobacco leaves. Inserts

788 show a close-up image of the association of NRAMP2-GFP with the various Golgi

789 subcompartments. Scale bars: 5 µm.

790 (E) and (F) pixel intensity profiles of GFP (green), RFP (magenta) and CFP (blue) were

791 measured along a line spanning the sub-compartments magnified in the close-up inserts

792 shown in (C) and (D), respectively.

793

794 Figure 5. NRAMP2 complements the smf2∆ and mtm1∆ yeast mutants.

795 (A), (D) and (E) Growth tests of wild type (BY4741) and mutant yeast strains smf1∆ (A),

796 smf2∆ (A,E), mtm1∆ (D) and pmr1∆ (E) transformed with the pYPGE15 empty vector (E.V.)

797 or the pYPGE15/NRAMP2 construct (NRAMP2). 5-fold serial dilutions were plated on SD-

798 Ura supplemented or not (control) with the amount of EGTA indicated in the figure panels

799 (A, D) or 2 mM MnSO4 (E) and incubated at 30ºC for 3 days.

800 (A) NRAMP2 restores smf2∆ mutant tolerance to Mn deficiency.

801 (B) Mn content measured by MP-AES in yeast strains grown in liquid SD -URA for 24 hours.

802 Mean ± SD (n-3). Asterisks indicate values significantly different from those of wild type

803 (Student’s t test, * P-value < 0.01).

804 (C) In gel SOD activity assay carried out on proteins extracted from yeast strains grown as in

805 (B).

806 (D) NRAMP2 restores mtm1∆ mutant tolerance to Mn deficiency.

807 (E) NRAMP2 fails to rescue pmr1∆ mutant sensitivity to excess Mn and does not reduce

808 wild-type or smf2∆ tolerance to excess Mn.

809

810 Figure 6. Photosynthetic activity is impaired in nramp2-3.

811 Maximum PSII efficiency (Fv/Fm) measured on rosette leaves of wild-type, nramp2-3 and

812 two complemented lines grown for 4 weeks in hydroponic conditions in the presence of Mn

26

813 (+Mn) or 1 week with Mn and transferred to medium without Mn for 3 additional weeks (-

814 Mn). Mean ± SD (n = 10 leaves from 10 plants). Asterisks indicate values significantly

815 different from those of wild type (Student’s t test, * P-value < 0.05, ** P-value < 0.01).

816

817 Figure 7. Vacuoles and chloroplasts of the nramp2-3 mutant contain less Mn than wild type.

818 (A) to (C) Mn content was measured on isolated protoplasts (A), chloroplasts (B) or vacuoles

819 (C) in wild-type, nramp2-3 and the OxNR2 #3 complemented line grown in hydroponic

820 cultures for one week in the presence of Mn followed by three additional weeks in the

821 absence of Mn. All panels share color code legends. Metal concentration was determined by

822 MP-AES and is expressed as ng per 106 protoplasts or organelles. Data are from one

823 representative experiment (n = 4 plants). Mean ± SD. Asterisks indicate values significantly

824 different from those of wild type (Student’s t test, * P-value < 0.01).

825

826 Figure 8. nramp2-3 displays high levels of oxidative stress.

827 (A) and (B) Plants of the indicated genotypes were cultivated in hydroponic conditions for 4

828 weeks in the presence of Mn (+Mn) or 1 week with Mn and transferred to medium without

829 Mn for 3 additional weeks (-Mn). (A) In-gel SOD activity assay performed using total

830 proteins extracted from leaves. (B) Detection of superoxide ion production in Mn replete or

831 Mn-starved leaves by staining with nitroblue tetrazolium (NBT).

832 (C) NRAMP2 rescues the sensitivity of the yeast sod1∆ mutant to Mn deficiency. 5-fold serial

833 dilutions were plated on SD-Ura supplemented with 1 mM EGTA (right) or not (Control,

834 left). Arrows indicate the growth rescue of smf2∆ by NRAMP2.

835

836 Figure 9. nramp2-3 shows cell wall permeability defects.

837 Plants of the indicated genotypes were grown in hydroponic conditions for 3 weeks in the

838 presence of Mn followed by an additional week in the presence (+ Mn) or absence of Mn (-

839 Mn).

840 (A) Permeability of the leaf surface was examined by staining entire leaves with Toluidine

841 blue for 10 min. Inserts show a closeup view of the TB-stained spots on the leaves.

842 (B) Cutin load per surface unit of leaf measured by GC-MS. Mean ± SD (n= 4 leaves from 4

843 plants). Values that are not significantly different are noted with the same letter (ANOVA, α

844 risk 5%, Supplemental File 1).

845

846 Figure 10. Hypothetical model of the function of NRAMP2 in cellular Mn distribution.

27

847 When NRAMP2 is nearly absent (nramp2 knockdown mutants), plants are hypersensitive to

848 Mn deficiency and PSII activity is markedly impaired. NRAMP2 is a transporter localized in

849 the TGN membrane, where it likely exports Mn from the TGN lumen to the cytosol. In doing

850 so, NRAMP2 builds up a pool of Mn (red circle) that, based on the pleiotropic effects of the

851 NRAMP2 mutation, is used to feed an array of downstream organelles: vacuoles and

852 chloroplasts, since both display decreased Mn content in nramp2-3, and mitochondria based

853 on the ability of NRAMP2 to complement yeast mtm1∆. Combining nramp2-3 with nramp3-2

854 and nramp4-2 mutations suggests that NRAMP2 and NRAMP3/NRAMP4 act in concert to

855 provide Mn to the PSII, NRAMP2 being epistatic to the other two.

856

857

858 REFERENCES 859 Agorio, A., Giraudat, J., Bianchi, M.W., Marion, J., Espagne, C., 860 Castaings, L., Lelievre, F., Curie, C., Thomine, S., and Merlot, S.861 (2017). Phosphatidylinositol 3-phosphate-binding protein AtPH1862 controls the localization of the metal transporter NRAMP1 in863 Arabidopsis. Proc Natl Acad Sci U S A 114, E3354-E3363.864 Allen, M.D., Kropat, J., Tottey, S., Del Campo, J.A., and Merchant, S.S.865 (2007). Manganese deficiency in Chlamydomonas results in loss of866 photosystem II and MnSOD function, sensitivity to peroxides, and867 secondary phosphorus and iron deficiency. Plant Physiol 143, 263-277.868 Barberon, M., Vermeer, J.E., De Bellis, D., Wang, P., Naseer, S., Andersen,869 T.G., Humbel, B.M., Nawrath, C., Takano, J., Salt, D.E., and Geldner,870 N. (2016). Adaptation of root function by nutrient-induced plasticity871 of endodermal differentiation. Cell 164, 447-459.872 Bolte, S., and Cordelieres, F.P. (2006). A guided tour into subcellular873 colocalization analysis in light microscopy. J Microsc 224, 213-232.874 Brandizzi, F., Frangne, N., Marc-Martin, S., Hawes, C., Neuhaus, J.M., and875 Paris, N. (2002). The destination for single-pass membrane proteins876 is influenced markedly by the length of the hydrophobic domain. Plant877 Cell 14, 1077-1092.878 Cailliatte, R., Lapeyre, B., Briat, J.F., Mari, S., and Curie, C. (2009).879 The NRAMP6 metal transporter contributes to cadmium toxicity. Biochem880 J 422, 217-228.881 Cailliatte, R., Schikora, A., Briat, J.F., Mari, S., and Curie, C. (2010).882 High-affinity manganese uptake by the metal transporter NRAMP1 is883 essential for Arabidopsis growth in low manganese conditions. The884 Plant cell 22, 904-917.885 Castaings, L., Caquot, A., Loubet, S., and Curie, C. (2016). The high-886 affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron887 under Sufficient Metal Provision. Sci Rep 6, 37222.888 Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for889 Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant890 J. 16, 735-743.891 Cohen, A., Nelson, H., and Nelson, N. (2000). The Family of SMF Metal Ion892 Transporters in Yeast Cells. J. Biol. Chem. 275, 33388-33394.893 Culotta, V.C., and Daly, M.J. (2013). Manganese complexes: diverse894 metabolic routes to oxidative stress resistance in prokaryotes and895 yeast. Antioxid Redox Signal 19, 933-944.

28

896 Curie, C., Alonso, J.M., Le Jean, M., Ecker, J.R., and Briat, J.F. (2000).897 Involvement of NRAMP1 from Arabidopsis thaliana in iron transport.898 Biochem. J. 347, 749-755.899 del Rio, L.A., Sandalio, L.M., Altomare, D.A., and Zilinskas, B.A. (2003).900 Mitochondrial and peroxisomal manganese superoxide dismutase:901 differential expression during leaf senescence. J Exp Bot 54, 923-902 933.903 Delhaize, E., Gruber, B.D., Pittman, J.K., White, R.G., Leung, H., Miao,904 Y., Jiang, L., Ryan, P.R., and Richardson, A.E. (2007). A role for905 the AtMTP11 gene of Arabidopsis in manganese transport and tolerance.906 Plant J 51, 198-210.907 Dinneny, J.R., Long, T.A., Wang, J.Y., Jung, J.W., Mace, D., Pointer, S.,908 Barron, C., Brady, S.M., Schiefelbein, J., and Benfey, P.N. (2008).909 Cell identity mediates the response of Arabidopsis roots to abiotic910 stress. Science 320, 942-945.911 Divol, F., Couch, D., Conejero, G., Roschzttardtz, H., Mari, S., and Curie,912 C. (2013). The Arabidopsis YELLOW STRIPE LIKE4 and 6 transporters913 control iron release from the chloroplast. The Plant cell 25, 1040-914 1055.915 Domergue, F., Vishwanath, S.J., Joubes, J., Ono, J., Lee, J.A., Bourdon,916 M., Alhattab, R., Lowe, C., Pascal, S., Lessire, R., and Rowland, O.917 (2010). Three Arabidopsis fatty acyl-coenzyme A reductases, FAR1,918 FAR4, and FAR5, generate primary fatty alcohols associated with919 suberin deposition. Plant Physiol 153, 1539-1554.920 Drakakaki, G., van de Ven, W., Pan, S., Miao, Y., Wang, J., Keinath, N.F.,921 Weatherly, B., Jiang, L., Schumacher, K., Hicks, G., and Raikhel, N.922 (2012). Isolation and proteomic analysis of the SYP61 compartment923 reveal its role in exocytic trafficking in Arabidopsis. Cell Res 22,924 413-424.925 Eroglu, S., Meier, B., von Wiren, N., and Peiter, E. (2016). The Vacuolar926 Manganese Transporter MTP8 Determines Tolerance to Iron Deficiency-927 Induced Chlorosis in Arabidopsis. Plant Physiol 170, 1030-1045.928 Foresti, O., Gershlick, D.C., Bottanelli, F., Hummel, E., Hawes, C., and929 Denecke, J. (2010). A recycling-defective vacuolar sorting receptor930 reveals an intermediate compartment situated between prevacuoles and931 vacuoles in tobacco. Plant Cell 22, 3992-4008.932 Garcia-Rodriguez, N., Diaz de la Loza Mdel, C., Andreson, B., Monje-Casas,933 F., Rothstein, R., and Wellinger, R.E. (2012). Impaired manganese934 metabolism causes mitotic misregulation. J Biol Chem 287, 18717-935 18729.936 Garcia-Rodriguez, N., Manzano-Lopez, J., Munoz-Bravo, M., Fernandez-Garcia,937 E., Muniz, M., and Wellinger, R.E. (2015). Manganese redistribution938 by calcium-stimulated vesicle trafficking bypasses the need for P-939 type ATPase function. J Biol Chem 290, 9335-9347.940 Geldner, N., Denervaud-Tendon, V., Hyman, D.L., Mayer, U., Stierhof, Y.D.,941 and Chory, J. (2009). Rapid, combinatorial analysis of membrane942 compartments in intact plants with a multicolor marker set. Plant J943 59, 169-178.944 Hebbern, C.A., Laursen, K.H., Ladegaard, A.H., Schmidt, S.B., Pedas, P.,945 Bruhn, D., Schjoerring, J.K., Wulfsohn, D., and Husted, S. (2009).946 Latent manganese deficiency increases transpiration in barley947 (Hordeum vulgare). Physiol Plant 135, 307-316.948 Hoffmann-Benning, S., and Kende, H. (1994). Cuticle Biosynthesis in Rapidly949 Growing Internodes of Deepwater Rice. Plant Physiol 104, 719-723.950 Ishimaru, Y., Takahashi, R., Bashir, K., Shimo, H., Senoura, T., Sugimoto,951 K., Ono, K., Yano, M., Ishikawa, S., Arao, T., Nakanishi, H., and952 Nishizawa, N.K. (2012). Characterizing the role of rice NRAMP5 in953 Manganese, Iron and Cadmium Transport. Sci Rep 2, 286.954 Kim, S.A., Punshon, T., Lanzirotti, A., Li, L., Alonso, J.M., Ecker, J.R.,955 Kaplan, J., and Guerinot, M.L. (2006). Localization of iron in

29

956 Arabidopsis seed requires the vacuolar membrane transporter VIT1.957 Science 314, 1295-1298.958 Lanquar, V., Ramos, M.S., Lelievre, F., Barbier-Brygoo, H., Krieger-959 Liszkay, A., Kramer, U., and Thomine, S. (2010). Export of vacuolar960 manganese by AtNRAMP3 and AtNRAMP4 is required for optimal961 photosynthesis and growth under manganese deficiency. Plant Physiol962 152, 1986-1999.963 Lanquar, V., Lelievre, F., Bolte, S., Hames, C., Alcon, C., Neumann, D.,964 Vansuyt, G., Curie, C., Schroder, A., Kramer, U., Barbier-Brygoo, H.,965 and Thomine, S. (2005). Mobilization of vacuolar iron by AtNRAMP3 and966 AtNRAMP4 is essential for seed germination on low iron. Embo J 24,967 4041-4051.968 Lin, Y.T., Hoang, H., Hsieh, S.I., Rangel, N., Foster, A.L., Sampayo, J.N.,969 Lithgow, G.J., and Srinivasan, C. (2006). Manganous ion970 supplementation accelerates wild type development, enhances stress971 resistance, and rescues the life span of a short-lived Caenorhabditis972 elegans mutant. Free Radic Biol Med 40, 1185-1193.973 Luk, E., Carroll, M., Baker, M., and Culotta, V.C. (2003). Manganese974 activation of superoxide dismutase 2 in Saccharomyces cerevisiae975 requires MTM1, a member of the mitochondrial carrier family. Proc976 Natl Acad Sci U S A 100, 10353-10357.977 Luk, E.E., and Culotta, V.C. (2001). Manganese Superoxide Dismutase in978 Saccharomyces cerevisiae Acquires Its Metal Co-factor through a979 Pathway Involving the Nramp Metal Transporter, Smf2p. J Biol Chem980 276, 47556-47562.981 Marschner, H., Romheld, V., and Kissel, M. (1986). Different strategies in982 higher plants in mobilization and uptake of iron. J. Plant Nutr. 9,983 3-7.984 Martiniere, A., Bassil, E., Jublanc, E., Alcon, C., Reguera, M., Sentenac,985 H., Blumwald, E., and Paris, N. (2013). In vivo intracellular pH986 measurements in tobacco and Arabidopsis reveal an unexpected pH987 gradient in the endomembrane system. Plant Cell 25, 4028-4043.988 McNaughton, R.L., Reddi, A.R., Clement, M.H., Sharma, A., Barnese, K.,989 Rosenfeld, L., Gralla, E.B., Valentine, J.S., Culotta, V.C., and990 Hoffman, B.M. (2010). Probing in vivo Mn2+ speciation and oxidative991 stress resistance in yeast cells with electron-nuclear double992 resonance spectroscopy. Proc Natl Acad Sci U S A 107, 15335-15339.993 Mills, R.F., Doherty, M.L., Lopez-Marques, R.L., Weimar, T., Dupree, P.,994 Palmgren, M.G., Pittman, J.K., and Williams, L.E. (2008). ECA3, a995 Golgi-localized P2A-type ATPase, plays a crucial role in manganese996 nutrition in Arabidopsis. Plant Physiol 146, 116-128.997 Milner, M.J., Seamon, J., Craft, E., and Kochian, L.V. (2013). Transport998 properties of members of the ZIP family in plants and their role in999 Zn and Mn homeostasis. J Exp Bot 64, 369-381.

1000 Momonoi, K., Yoshida, K., Mano, S., Takahashi, H., Nakamori, C., Shoji, K.,1001 Nitta, A., and Nishimura, M. (2009). A vacuolar iron transporter in1002 tulip, TgVit1, is responsible for blue coloration in petal cells1003 through iron accumulation. Plant J 59, 437-447.1004 Nelson, N., Sacher, A., and Nelson, H. (2002). The significance of1005 molecular slips in transport systems. Nat Rev Mol Cell Biol 3, 876-1006 881.1007 Nevo, Y., and Nelson, N. (2006). The NRAMP family of metal-ion1008 transporters. Biochim Biophys Acta 1763, 609-620.1009 Nickelsen, J., and Rengstl, B. (2013). Photosystem II assembly: from1010 cyanobacteria to plants. Annu Rev Plant Biol 64, 609-635.1011 Pedas, P., Schiller Stokholm, M., Hegelund, J.N., Ladegard, A.H.,1012 Schjoerring, J.K., and Husted, S. (2014). Golgi localized barley MTP81013 proteins facilitate Mn transport. PLoS One 9, e113759.1014 Peiter, E., Montanini, B., Gobert, A., Pedas, P., Husted, S., Maathuis,1015 F.J., Blaudez, D., Chalot, M., and Sanders, D. (2007). A secretory

30

1016 pathway-localized cation diffusion facilitator confers plant1017 manganese tolerance. Proc Natl Acad Sci U S A 104, 8532-8537.1018 Pittman, J.K., Shigaki, T., Marshall, J.L., Morris, J.L., Cheng, N.H., and1019 Hirschi, K.D. (2004). Functional and regulatory analysis of the1020 Arabidopsis thaliana CAX2 cation transporter. Plant Mol Biol 56, 959-1021 971.1022 Portnoy, M.E., Liu, X.F., and Culotta, V.C. (2000). Saccharomyces1023 cerevisiae expresses three functionally distinct homologues of the1024 nramp family of metal transporters. Mol. Cell. Biol. 20, 7893-7902.1025 Ravet, K., Touraine, B., Kim, S.A., Cellier, F., Thomine, S., Guerinot,1026 M.L., Briat, J.F., and Gaymard, F. (2009). Post-translational1027 regulation of AtFER2 ferritin in response to intracellular iron1028 trafficking during fruit development in Arabidopsis. Mol Plant 2,1029 1095-1106.1030 Roth, J.A. (2006). Homeostatic and toxic mechanisms regulating manganese1031 uptake, retention, and elimination. Biol Res 39, 45-57.1032 Rudolph, H.K., Antebi, A., Fink, G.R., Buckley, C.M., Dorman, T.E.,1033 LeVitre, J., Davidow, L.S., Ma

Documents

Intracellular Distribution of Manganese by the Trans-Golgi … · 2017. 11. 27. · Intracellular Distribution of Manganese by the TransGolgi Network - Transporter NRAMP2 is Critical